Peptidomimetic macrocycles with improved properties

ABSTRACT

The present invention provides biologically active peptidomimetic macrocycles with improved properties relative to their corresponding polypeptides. The invention additionally provides methods of preparing and using such macrocycles, for example in therapeutic applications.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/117,508, filed Nov. 24, 2008, which is incorporated herein in itsentirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 16, 2010, is named35224742.txt and is 72,471 bytes in size.

BACKGROUND OF THE INVENTION

Recombinant or synthetically produced polypeptides have importantapplications as pharmaceuticals. Polypeptides such as short peptides,however, often suffer from poor metabolic stability, poor cellpenetrability, and promiscuous binding due to conformationalflexibility. Various approaches to stabilizing helical peptides havebeen tried, for example by using intramolecular crosslinkers to maintainthe peptide in a desired configuration by introducing disulfide bonds,amide bonds, or carbon-carbon bonds to link amino acid side chains. See,e.g., Jackson et al. (1991), J. Am. Chem. Soc. 113:9391-9392; Phelan etal. (1997), J. Am. Chem. Soc. 119:455-460; Taylor (2002), Biopolymers66: 49-75; Brunel et al. (2005), Chem. Commun. (20):2552-2554; Hiroshigeet al. (1995), J. Am. Chem. Soc. 117: 11590-11591; Blackwell et al.(1998), Angew. Chem. Int. Ed. 37:3281-3284; Schafineister et al. (2000),J. Am. Chem. Soc. 122:5891-5892; Walensky et al. (2004), Science305:1466-1470; Bernal et al. (2007), J. Am. Chem. Soc. 129:2456-2457;United States Patent Application 2005/0250680, filed Nov. 5, 2004; U.S.Pat. No. 7,192,713 B1 (Verdine et al); U.S. patent application Ser. No.11/957,325 filed Dec. 14, 2007; U.S. patent application Ser. No.12/037,041 filed Feb. 25, 2008 and U.S. Pat. No. 5,811,515, the contentsof which patents and publications are incorporated herein by reference.There remains a significant need for therapeutic and pharmaceuticallyuseful polypeptides with improved biological properties such as improvedin vivo half-lives, efficacy at lower doses or reduced frequency ofadministration.

SUMMARY OF THE INVENTION

The present invention addresses these and other needs. In one aspect,the present invention provides helical peptidomimetic macrocycles withimproved pharmacokinetic properties relative to their correspondingnon-cross-linked counterparts.

For example, the present invention provides a method of increasing thein vivo half-life of a helical polypeptide by installing one or morecross-links. In some embodiments of the method, the in vivo half-life ofsaid polypeptide is increased on average at least 50-fold relative to acorresponding polypeptide lacking said cross-links. In other embodimentsof the method, the in vivo half-life of said polypeptide is increased atleast 100-fold, 150-fold or 200-fold relative to a correspondingpolypeptide lacking said cross-links. In some embodiments, thepolypeptide is selected such that the apparent serum binding affinity(Kd*) of the crosslinked polypeptide is 1, 3, 10, 70 micromolar orgreater. In other embodiments, the Kd* of the crosslinked polypeptide is1 to 10, 70, or 700 micromolar. In other embodiments, the crosslinkedpolypeptides is selected such that it possesses an estimated freefraction in human blood of between 0.1 and 50%, or between 0.15 and 10%.In some embodiments, the polypeptide is selected such that the %helicity of the crosslinked polypeptide is greater than 25%, 50% or 75%at room temperature under aqueous conditions. In other embodiments, the% helicity of the crosslinked polypeptide is increased at least 2-fold,5-fold or 10-fold relative to a corresponding polypeptide lacking saidcross-links.

In some embodiments of the method, said polypeptide contains onecrosslink. In other embodiments of the method, said polypeptide containstwo cross-links.

In some embodiments of the method, one crosslink connects two α-carbonatoms. In other embodiments of the method, one α-carbon atom to whichone crosslink is attached is substituted with a substituent of formulaR—. In another embodiment of the method, two α-carbon atoms to which onecrosslink is attached are substituted with independent substituents offormula R—.

In one embodiment of the methods of the invention, R— is alkyl. Forexample, R— is methyl. Alternatively, R— and any portion of onecrosslink taken together can form a cyclic structure. In anotherembodiment of the method, one crosslink is formed of consecutivecarbon-carbon bonds. For example, one crosslink may comprise at least 8,9, 10, 11, or 12 consecutive bonds. In other embodiments, one crosslinkmay comprise at least 7, 8, 9, 10, or 11 carbon atoms.

In another embodiment of the method, the crosslinked polypeptidecomprises an α-helical domain of a BCL-2 family member. For example, thecrosslinked polypeptide comprises a BH3 domain. In other embodiments,the crosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90%or 95% of any of the sequences in Tables 1, 2, 3 and 4.

In some embodiments of the method, the crosslinked polypeptidepenetrates cell membranes by an energy-dependent process and binds to anintracellular target.

In other embodiments, the present invention provides a helicalpolypeptide comprising one or more cross-links, wherein the cross-linkedhelical polypeptide has an in vivo half-life greater than 360 minutes.In other embodiments, the in vivo half-life of said polypeptide isgreater than 500 minutes or 1,000 minutes. In another embodiment, the invivo half-life of said polypeptide is between 500-5,000 minutes.

In some embodiments, said helical polypeptide contains one crosslink. Inother embodiments, said helical polypeptide contains two cross-links.

In some embodiments, one crosslink connects two α-carbon atoms. In otherembodiments, one α-carbon atom to which one crosslink is attached issubstituted with a substituent of formula R—. In another embodiment, twoα-carbon atoms to which one crosslink is attached are substituted withindependent substituents of formula R—.

In one embodiment of the invention, R— is alkyl. For example, R— ismethyl. Alternatively, R— and any portion of one crosslink takentogether can form a cyclic structure. In another embodiment, onecrosslink is formed of consecutive carbon-carbon bonds. For example, onecrosslink may comprise at least 8, 9, 10, 11, or 12 consecutive bonds.In other embodiments, one crosslink may comprise at least 7, 8, 9, 10,or 11 carbon atoms.

In another embodiment, the crosslinked polypeptide comprises anα-helical domain of a BCL-2 family member. For example, the crosslinkedpolypeptide comprises a BH3 domain. In other embodiments, thecrosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90% or95% of any of the sequences in Tables 1, 2, 3 and 4.

In some embodiments, the crosslinked polypeptide penetrates cellmembranes by an energy-dependent process and binds to an intracellulartarget.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 describes sequences of peptidomimetic macrocycles of theinvention and sequences of the corresponding non-cross-linkedcounterparts (SEQ ID NOS 118-138, respectively, in order of appearance).

FIG. 2 lists the in vivo half-lives of peptidomimetic macrocycles of theinvention. Half-lives were measured in Sprague Dawley rats after oneintravenous bolus injection at 0.6 mg/mL or 2 mg/mL of each unlabeledpeptidomimetic macrocycle at a 3 mg/kg or 10 mg/kg dose, respectively.Three animals were used per compound and concentrations were determinedby mass-spectrometric analysis of blood levels (plasma).

FIGS. 3a-u illustrate blood plasma concentration curves for severalpeptidomimetic macrocycles of the invention.

FIG. 4 shows the apparent rat serum binding affinity and estimated freefraction in rat blood of peptidomimetic marocycles of the invention.

FIG. 5 illustrates the PK profile in rat and monkey for a peptidomimeticmacrocycle of the invention.

FIG. 6 illustrates actual and predicted PK properties such as clearancerates of peptidomimetic macrocycles of the invention in rodents andhigher species.

FIG. 7 shows the molar ellipticity at 222 nm and estimated % helicity ofpeptidomimetic marocycles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinkedpolypeptide” refers to a compound comprising a plurality of amino acidresidues joined by a plurality of peptide bonds and at least onemacrocycle-forming linker which forms a macrocycle between a firstnaturally-occurring or non-naturally-occurring amino acid residue (oranalog) and a second naturally-occurring or non-naturally-occurringamino acid residue (or analog) within the same molecule. Peptidomimeticmacrocycle include embodiments where the macrocycle-forming linkerconnects the α carbon of the first amino acid residue (or analog) to theα carbon of the second amino acid residue (or analog). Thepeptidomimetic macrocycles optionally include one or more non-peptidebonds between one or more amino acid residues and/or amino acid analogresidues, and optionally include one or more non-naturally-occurringamino acid residues or amino acid analog residues in addition to anywhich form the macrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a peptidomimetic macrocycleof the invention as measured by circular dichroism, NMR or anotherbiophysical measure, or resistance to proteolytic degradation in vitroor in vivo. Non-limiting examples of secondary structures contemplatedin this invention are α-helices, β-turns, and β-pleated sheets.

As used herein, the term “helical stability” refers to the maintenanceof α helical structure by a peptidomimetic macrocycle of the inventionas measured by circular dichroism or NMR. For example, in someembodiments, the peptidomimetic macrocycles of the invention exhibit atleast a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determinedby circular dichroism compared to a corresponding uncrosslinkedpolypeptide.

The term “α-amino acid” or simply “amino acid” refers to a moleculecontaining both an amino group and a carboxyl group bound to a carbonwhich is designated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The term “amino acid analog” or “non-natural amino acid” refers to amolecule which is structurally similar to an amino acid and which can besubstituted for an amino acid in the formation of a peptidomimeticmacrocycle. Amino acid analogs include, without limitation, compoundswhich are structurally identical to an amino acid, as defined herein,except for the inclusion of one or more additional methylene groupsbetween the amino and carboxyl group (e.g., α-amino β-carboxy acids), orfor the substitution of the amino or carboxy group by a similarlyreactive group (e.g., substitution of the primary amine with a secondaryor tertiary amine, or substitution or the carboxy group with an ester).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., a BH3 domain or thep53 MDM2 binding domain) without abolishing or substantially alteringits essential biological or biochemical activity (e.g., receptor bindingor activation). An “essential” amino acid residue is a residue that,when altered from the wild-type sequence of the polypeptide, results inabolishing or substantially abolishing the polypeptide's essentialbiological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a BH3 polypeptide, for example, ispreferably replaced with another amino acid residue from the same sidechain family. Other examples of acceptable substitutions aresubstitutions based on isosteric considerations (e.g. norleucine formethionine) or other properties (e.g. 2-thienylalanine forphenylalanine).

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol

when used as part of a molecular structure refers to a single bond or atrans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an α,α di-substituted aminoacid).

The term “α,α di-substituted amino” acid refers to a molecule or moietycontaining both an amino group and a carboxyl group bound to a carbon(the α-carbon) that is attached to two natural or non-natural amino acidside chains.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “macrocyclization reagent” or “macrocycle-forming reagent” asused herein refers to any reagent which may be used to prepare apeptidomimetic macrocycle of the invention by mediating the reactionbetween two reactive groups. Reactive groups may be, for example, anazide and alkyne, in which case macrocyclization reagents include,without limitation, Cu reagents such as reagents which provide areactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II)salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted insitu to an active Cu(I) reagent by the addition of a reducing agent suchas ascorbic acid or sodium ascorbate. Macrocyclization reagents mayadditionally include, for example, Ru reagents known in the art such asCp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which may provide areactive Ru(II) species. In other cases, the reactive groups areterminal olefins. In such embodiments, the macrocyclization reagents ormacrocycle-forming reagents are metathesis catalysts including, but notlimited to, stabilized, late transition metal carbene complex catalystssuch as Group VIII transition metal carbene catalysts. For example, suchcatalysts are Ru and Os metal centers having a +2 oxidation state, anelectron count of 16 and pentacoordinated. Additional catalysts aredisclosed in Grubbs et al., “Ring Closing Metathesis and RelatedProcesses in Organic Synthesis” Acc. Chem. Res. 1995, 28, 446-452, andU.S. Pat. No. 5,811,515. In yet other cases, the reactive groups arethiol groups. In such embodiments, the macrocyclization reagent is, forexample, a linker functionalized with two thiol-reactive groups such ashalogen groups.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃,—C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH₃, and—CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced witha—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, or any other real values ≧0 and ≦2 if thevariable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “on average” represents the mean value derived from performingat least three independent replicates for each data point.

The term “biological activity” encompasses structural and functionalproperties of a macrocycle of the invention. Biological activity is, forexample, structural stability, alpha-helicity, affinity for a target,resistance to proteolytic degradation, cell penetrability, intracellularstability, in vivo stability, or any combination thereof.

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

Biological Properties of the Peptidomimetic Macrocycles of the Invention

In one aspect, the invention provides a method of increasing the in vivohalf-life of a helical polypeptide by installing one or morecross-links. For example, the in vivo half-life of said polypeptide isincreased on average at least 50-fold relative to a correspondingpolypeptide lacking said cross-links. In other embodiments of themethod, the in vivo half-life of said polypeptide is increased at least100-fold, 150-fold or 200-fold relative to a corresponding polypeptidelacking said cross-links. In other embodiments, the present inventionprovides a helical polypeptide comprising one or more cross-links,wherein the cross-linked helical polypeptide has an in vivo half-lifegreater than 360 minutes. In other embodiments, the in vivo half-life ofsaid polypeptide is greater than 500 minutes or 1,000 minutes. Inanother embodiment, the in vivo half-life of said polypeptide is between500-5,000 minutes. In another embodiment, the in vivo half-life of saidpolypeptide is determined after intravenous administration.

In some embodiments, the polypeptide is selected such that the apparentserum binding affinity (Kd*) of the crosslinked polypeptide is 1, 3, 10,70 micromolar or greater. In other embodiments, the Kd* of thecrosslinked polypeptide is 1 to 10, 70, or 700 micromolar. In otherembodiments, the crosslinked polypeptides is selected such that itpossesses an estimated free fraction in human blood of between 0.1 and50%, or between 0.15 and 10%.

The present invention provides a method of identifying cross-linkedpolypeptides with the desired serum binding affinities, comprising thesteps of synthesizing analogs of the parent cross-linked polypeptide andperforming cellular assays in the absence of serum proteins and also inthe presence of two or more concentrations of serum, so as to determinethe apparent affinity of each cross-linked polypeptide for serumproteins and to calculate an EC₅₀ in whole blood by mathematicalextrapolation.

In one embodiment, the apparent Kd values for serum protein by EC50shift analysis is used to provide a simple and rapid means ofquantifying the propensity of experimental compounds to bind HSA andother serum proteins. A linear relationship exists between the apparentEC₅₀ in the presence of serum protein (EC′₅₀) and the amount of serumprotein added to an in vitro assay. This relationship is defined by thebinding affinity of the compound for serum proteins, expressed asK_(d)*. This term is an experimentally determined, apparent dissociationconstant that may result from the cumulative effects of multiple,experimentally indistinguishable, binding events. The form of thisrelationship is presented here in Eq. 1, and its derivation can be foundin Copeland et al, Biorg. Med. Chem. Lett. 2004, 14:2309-2312, thecontents of which are incorporated herein by reference.

$\begin{matrix}{{EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}} & (1)\end{matrix}$

A significant proportion of serum protein binding can be ascribed todrug interactions with serum albumin, due to the very high concentrationof this protein in serum (35-50 g/L or 530-758 μM). To calculate theK_(d) value for these compounds we have assumed that the shift in EC₅₀upon protein addition can be ascribed fully to the serum albumin presentin the added serum, where P is 700 μM for 100% serum, P is 70 μM for 10%serum, etc. We further make the simplifying assumption that all of thecompounds bind serum albumin with a 1:1 stoichiometry, so that the termn in Eq. (1) is fixed at unity. With these parameters in place wecalculate the K_(d)* value for each stapled peptide from the changes inEC₅₀ values with increasing serum (and serum protein) concentrations bynonlinear regression analysis using Mathematica 4.1 (Wolfram Research,Inc., www.wolfram.com). The free fraction in blood is estimated per thefollowing equation, where [serum albumin]_(total) is set at 700 μM, asderived by Trainor, Expert Opin. Drug Disc., 2007, 2(1):51-64, thecontents of which are incorporated herein by reference. The formulabelow shows an embodiment where the serum albumin is human serumalbumin.

$\begin{matrix}{{FreeFraction} = \frac{K_{d}^{*}}{K_{d}^{*} + \lbrack{HSA}\rbrack_{total}}} & (2)\end{matrix}$

In one embodiment, the improved biological activity is measured asincreased cell penetrability or an increased ability to induceapoptosis. In yet other embodiments, the biological activity is measuredas the percentage of the number of cells killed in an in vitro assay inwhich cultured cells are exposed to an effective concentration of saidpolypeptide.

In some embodiments, the polypeptide is selected such that the percenthelicity of the crosslinked polypeptide is greater than 25%, 50% or 75%at room temperature under aqueous conditions. In other embodiments, thepercent helicity at room temperature under aqueous conditions of thecrosslinked polypeptide is increased at least 2-fold, 5-fold or 10-foldrelative to a corresponding polypeptide lacking said cross-links.

Design of the Peptidomimetic Macrocycles of the Invention

Any protein or polypeptide with a known primary amino acid sequencewhich contains a helical structure believed to impart biologicalactivity is the subject of the present invention. For example, thesequence of the polypeptide can be analyzed and amino acid analogscontaining groups reactive with macrocyclization reagents can besubstituted at the appropriate positions. The appropriate positions aredetermined by ascertaining which molecular surface(s) of the secondarystructure is (are) required for biological activity and, therefore,across which other surface(s) the macrocycle forming linkers of theinvention can form a macrocycle without sterically blocking thesurface(s) required for biological activity. Such determinations aremade using methods such as X-ray crystallography of complexes betweenthe secondary structure and a natural binding partner to visualizeresidues (and surfaces) critical for activity; by sequential mutagenesisof residues in the secondary structure to functionally identify residues(and surfaces) critical for activity; or by other methods. By suchdeterminations, the appropriate amino acids are substituted with theamino acids analogs and macrocycle-forming linkers of the invention. Forexample, for an α-helical secondary structure, one surface of the helix(e.g., a molecular surface extending longitudinally along the axis ofthe helix and radially 45-135° about the axis of the helix) may berequired to make contact with another biomolecule in vivo or in vitrofor biological activity. In such a case, a macrocycle-forming linker isdesigned to link two α-carbons of the helix while extendinglongitudinally along the surface of the helix in the portion of thatsurface not directly required for activity.

In some embodiments of the invention, the peptide sequence is derivedfrom the BCL-2 family of proteins. The BCL-2 family is defined by thepresence of up to four conserved BCL-2 homology (BH) domains designatedBH1, BH2, BH3, and BH4, all of which include α-helical segments(Chittenden et al. (1995), EMBO 14:5589; Wang et al. (1996), Genes Dev.10:2859). Anti-apoptotic proteins, such as BCL-2 and BCL-X_(L), displaysequence conservation in all BH domains. Pro-apoptotic proteins aredivided into “multidomain” family members (e.g., BAK, BAX), whichpossess homology in the BH1, BH2, and BH3 domains, and “BH3-domain only”family members (e.g., BID, BAD, BIM, BIK, NOXA, PUMA), that containsequence homology exclusively in the BH3 amphipathic α-helical segment.BCL-2 family members have the capacity to form homo- and heterodimers,suggesting that competitive binding and the ratio between pro- andanti-apoptotic protein levels dictates susceptibility to death stimuli.Anti-apoptotic proteins function to protect cells from pro-apoptoticexcess, i.e., excessive programmed cell death. Additional “security”measures include regulating transcription of pro-apoptotic proteins andmaintaining them as inactive conformers, requiring either proteolyticactivation, dephosphorylation, or ligand-induced conformational changeto activate pro-death functions. In certain cell types, death signalsreceived at the plasma membrane trigger apoptosis via a mitochondrialpathway. The mitochondria can serve as a gatekeeper of cell death bysequestering cytochrome c, a critical component of a cytosolic complexwhich activates caspase 9, leading to fatal downstream proteolyticevents. Multidomain proteins such as BCL-2/BCL-X_(L) and BAK/BAX playdueling roles of guardian and executioner at the mitochondrial membrane,with their activities further regulated by upstream BH3-only members ofthe BCL-2 family. For example, BID is a member of the BH3-domain onlyfamily of pro-apoptotic proteins, and transmits death signals receivedat the plasma membrane to effector pro-apoptotic proteins at themitochondrial membrane. BID has the capability of interacting with bothpro- and anti-apoptotic proteins, and upon activation by caspase 8,triggers cytochrome c release and mitochondrial apoptosis. Deletion andmutagenesis studies determined that the amphipathic α-helical BH3segment of pro-apoptotic family members may function as a death domainand thus may represent a critical structural motif for interacting withmultidomain apoptotic proteins. Structural studies have shown that theBH3 helix can interact with anti-apoptotic proteins by inserting into ahydrophobic groove formed by the interface of BH1, 2 and 3 domains.Activated BID can be bound and sequestered by anti-apoptotic proteins(e.g., BCL-2 and BCL-X_(L)) and can trigger activation of thepro-apoptotic proteins BAX and BAK, leading to cytochrome c release anda mitochondrial apoptosis program. BAD is also a BH3-domain onlypro-apoptotic family member whose expression triggers the activation ofBAX/BAK. In contrast to BID, however, BAD displays preferential bindingto anti-apoptotic family members, BCL-2 and BCL-X_(L). Whereas the BADBH3 domain exhibits high affinity binding to BCL-2, BAD BH3 peptide isunable to activate cytochrome c release from mitochondria in vitro,suggesting that BAD is not a direct activator of BAX/BAK. Mitochondriathat over-express BCL-2 are resistant to BID-induced cytochrome crelease, but co-treatment with BAD can restore BID sensitivity.Induction of mitochondrial apoptosis by BAD appears to result fromeither: (1) displacement of BAX/BAK activators, such as BID and BID-likeproteins, from the BCL-2/BCL-XL binding pocket, or (2) selectiveoccupation of the BCL-2/BCL-XL binding pocket by BAD to preventsequestration of BID-like proteins by anti-apoptotic proteins. Thus, twoclasses of BH3-domain only proteins have emerged, BID-like proteins thatdirectly activate mitochondrial apoptosis, and BAD-like proteins, thathave the capacity to sensitize mitochondria to BID-like pro-apoptoticsby occupying the binding pockets of multidomain anti-apoptotic proteins.Various α-helical domains of BCL-2 family member proteins amendable tothe methodology disclosed herein have been disclosed (Walensky et al.(2004), Science 305:1466; and Walensky et al., U.S. Patent PublicationNo. 2005/0250680, the entire disclosures of which are incorporatedherein by reference).

In other embodiments, the peptide sequence is derived from the tumorsuppressor p53 protein which binds to the oncogene protein MDM2. TheMDM2 binding site is localized within a region of the p53 tumorsuppressor that forms an α helix. In U.S. Pat. No. 7,083,983, the entirecontents of which are incorporated herein by reference, Lane et al.disclose that the region of p53 responsible for binding to MDM2 isrepresented approximately by amino acids 13-31 (PLSQETFSDLWKLLPENNV)(SEQ ID NO: 1) of mature human P53 protein. Other modified sequencesdisclosed by Lane are also contemplated in the instant invention.Furthermore, the interaction of p53 and MDM2 has been discussed by Shairet al. (1997), Chem. & Biol. 4:791, the entire contents of which areincorporated herein by reference, and mutations in the p53 gene havebeen identified in virtually half of all reported cancer cases. Asstresses are imposed on a cell, p53 is believed to orchestrate aresponse that leads to either cell-cycle arrest and DNA repair, orprogrammed cell death. As well as mutations in the p53 gene that alterthe function of the p53 protein directly, p53 can be altered by changesin MDM2. The MDM2 protein has been shown to bind to p53 and disrupttranscriptional activation by associating with the transactivationdomain of p53. For example, an 11 amino-acid peptide derived from thetransactivation domain of p53 forms an amphipathic α-helix of 2.5 turnsthat inserts into the MDM2 crevice. Thus, in some embodiments, novelα-helix structures generated by the method of the present invention areengineered to generate structures that bind tightly to the helixacceptor and disrupt native protein-protein interactions. Thesestructures are then screened using high throughput techniques toidentify optimal small molecule peptides. The novel structures thatdisrupt the MDM2 interaction are useful for many applications,including, but not limited to, control of soft tissue sarcomas (whichover-expresses MDM2 in the presence of wild type p53). These cancers arethen, in some embodiments, held in check with small molecules thatintercept MDM2, thereby preventing suppression of p53. Additionally, insome embodiments, small molecules disrupters of MDM2-p53 interactionsare used as adjuvant therapy to help control and modulate the extent ofthe p53 dependent apoptosis response in conventional chemotherapy.

A non-limiting exemplary list of suitable peptide sequences for use inthe present invention is given below:

TABLE 1 Name Sequence (bold = SEQ Cross-linked Sequence  SEQcritical residues) ID NO: ( X = x-link residue) ID NO: BH3 peptidesBID-BH3 QEDIIRNIARHLAQVGDSMDRSIPP 2 QEDIIRNIARHLA X VGD X MDRSIPP 25BIM-BH3 DNRPEIWIAQELRRIGDEFNAYYAR 3 DNRPEIWIAQELR X IGD X FNAYYAR 26BAD-BH3 NLWAAQRYGRELRRMSDEFVDSFKK 4 NLWAAQRYGRELR X MSD X FVDSFKK 27PUMA-BH3 EEQWAREIGAQLRRMADDLNAQYER 5 EEQWAREIGAQLR X MAD X LNAQYER 28Hrk-BH3 RSSAAQLTAARLKALGDELHQRTM 6 RSSAAQLTAARLK X LGD X LHQRTM 29NOXAA-BH3 AELPPEFAAQLRKIGDKVYCTW 7 AELPPEFAAQLR X IGD X VYCTW 30NOXAB-BH3 VPADLKDECAQLRRIGDKVNLRQKL 8 VPADLKDECAQLR X IGD X VNLRQKL 31BMF-BH3 QHRAEVQIARKLQCIADQFHRLHT 9 QHRAEVQIARKLQ X IAD X FHRLHT 32BLK-BH3 SSAAQLTAARLKALGDELHQRT 10 SSAAQLTAARLK X LGD X LHQRT 33 BIK-BH3CMEGSDALALRLACIGDEMDVSLRA 11 CMEGSDALALRLA X IGD X MDVSLRA 34 Bnip3DIERRKEVESILKKNSDWIWDWSS 12 DIERRKEVESILK X NSD X IWDWSS 35 BOK-BH3GRLAEVCAVLLRLGDELEMIRP 13 GRLAEVCAVLL X LGD X LEMIRP 36 BAX-BH3PQDASTKKSECLKRIGDELDSNMEL 14 PQDASTKKSECLK X IGD

LDSNMEL 37 BAK-BH3 PSSTMGQVGRQLAIIGDDINRR 15 PSSTMGQVGRQLA X IGD X INRR38 BCL2L1-BH3 KQALREAGDEFELR 16 KQALR X AGD X FELR 39 BCL2-BH3LSPPVVHLALALRQAGDDFSRR 17 LSPPVVHLALALR X AGD X FSRR 40 BCL-XL-BH3EVIPMAAVKQALREAGDEFELRY 18 EVIPMAAVKQALR X AGD X FELRY 41 BCL-W-BH3PADPLHQAMRAAGDEFETRF 19 PADPLHQAMR X AGD X FETRF 42 MCL1-BH3ATSRKLETLRRVGDGVQRNHETA 20 ATSRKLETLR X VGD X VQRNHETA 43 MTD-BH3LAEVCTVLLRLGDELEQIR 21 LAEVCTVLL X LGD X LEQIR 44 MAP-1-BH3MTVGELSRALGHENGSLDP 22 MTVGELSRALG X ENG X LDP 45 NIX-BH3VVEGEKEVEALKKSADWVSDWS 23 VVEGEKEVEALK X SAD X VSDWS 46 4ICD(ERBB4)-SMARDPQRYLVIQGDDRMKL 24 SMARDPQRYLV X QGD X RMKL 47 BH3 Table 1 listshuman sequences which target the BH3 binding site and are implicated incancers, autoimmune disorders, metabolic diseases and other humandisease conditions.

TABLE 2 Name Sequence (bold = SEQ Cross-linked Sequence  SEQcritical residues) ID NO: ( X = x-link residue) ID NO: BH3 peptidesBID-BH3 QEDIIRNIARHLAQVGDSMDRSIPP 2 QEDIIRNI X RHL X QVGDSMDRSIPP 48BIM-BH3 DNRPEIWIAQELRRIGDEFNAYYAR 3 DNRPEIWI X QEL X RIGDEFNAYYAR 49BAD-BH3 NLWAAQRYGRELRRMSDEFVDSFKK 4 NLWAAQRY X REL X RMSDEFVDSFKK 50PUMA-BH3 EEQWAREIGAQLRRMADDLNAQYER 5 EEQWAREI X AQL X RMADDLNAQYER 51Hrk-BH3 RSSAAQLTAARLKALGDELHQRTM 6 RSSAAQLT X ARL X ALGDELHQRTM 52NOXAA-BH3 AELPPEFAAQLRKIGDKVYCTW 7 AELPPEF X AQL X KIGDKVYCTW 53NOXAB-BH3 VPADLKDECAQLRRIGDKVNLRQKL 8 VPADLKDE X AQL X RIGDKVNLRQKL 54BMF-BH3 QHRAEVQIARKLQCIADQFHRLHT 9 QHRAEVQI X RKL X CIADQFHRLHT 55BLK-BH3 SSAAQLTAARLKALGDELHQRT 10 SSAAQLT X ARL X ALGDELHQRT 56 BIK-BH3CMEGSDALALRLACIGDEMDVSLRA 11 CMEGSDAL X LRL X CIGDEMDVSLRA 57 Bnip3DIERRKEVESILKKNSDWIWDWSS 12 DIERRKEV X SIL X KNSDWIWDWSS 58 BOK-BH3GRLAEVCAVLLRLGDELEMIRP 13 GRLAEV X AVL X RLGDELEMIRP 59 BAX-BH3PQDASTKKSECLKRIGDELDSNMEL 14 PQDASTKK X ECL X RIGDELDSNMEL 60 BAK-BH3PSSTMGQVGRQLAIIGDDINRR 15 PSSTMGQV X RQL X IIGDDINRR 61 BCL2L1-BH3KQALREAGDEFELR 16 X QAL X EAGDEFELR 62 BCL2-BH3 LSPPVVHLALALRQAGDDFSRR17 LSPPVVHL X LAL X QAGDDFSRR 63 BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY 18EVIPMAAV X QAL X EAGDEFELRY 64 BCL-W-BH3 PADPLHQAMRAAGDEFETRF 19 PADPL XQAM X AAGDEFETRF 65 MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA 20 ATSRK X ETL XRVGDGVQRNHETA 66 MTD-BH3 LAEVCTVLLRLGDELEQIR 21 LAEV X TVL X RLGDELEQIR67 MAP-1-BH3 MTVGELSRALGHENGSLDP 22 MTVGEL X RAL X HENGSLDP 68 NIX-BH3VVEGEKEVEALKKSADWVSDWS 23 VVEGEKE X EAL X KSADWVSDWS 69 4ICD(ERBB4)-BH3SMARDPQRYLVIQGDDRMKL 24 SMARDP X RYL X IQGDDRMKL 70 Table 2 lists humansequences which target the BH3 binding site and are implicated incancers, autoimmune disorders, metabolic diseases and other humandisease conditions.

TABLE 3 Sequence (bold = SEQ Cross-linked Sequence SEQ Namecritical residues) ID NO: ( X = x-link residue) ID NO: P53 peptideshp53 peptide 1 LSQETFSDLWKLLPEN 71 LSQETFSD X WKLLPEX 72 hp53 peptide 2LSQETFSDLWKLLPEN 71 LSQE X FSDLWK X LPEN 73 hp53 peptide 3LSQETFSDLWKLLPEN 71 LSQ X TFSDLW X LLPEN 74 hp53 peptide 4LSQETFSDLWKLLPEN 71 LSQETF X DLWKLL X EN 75 hp53 peptide 5LSQETFSDLWKLLPEN 71 QSQQTF X NLWRLL X QN 76 Table 3 lists humansequences which target the p53 binding site of MDM2/X and are implicatedin cancers.

TABLE 4 Sequence (bold = SEQ Cross-linked Sequence SEQ Namecritical residues) ID NO: ( X = x-link residue) ID NO:GPCR peptide ligands Angiotensin II DRVYIHPF 77 DR X Y X HPF 83 BombesinEQRLGNQWAVGHLM 78 EQRLGN X WAVGHL X 84 Bradykinin RPPGFSPFR 79 RPP XFSPFR X 85 C5a ISHKDMQLGR 80 ISHKDM X LGR X 86 C3a ARASHLGLAR 81 ARASHLX LAR X 87 α-melanocyte  SYSMEHFRWGKPV 82 SYSM X HFRW X KPV 88stimulating hormone Table 4 lists sequences which target human Gprotein-coupled receptors and are implicated in numerous human diseaseconditions (Tyndall et al. (2005), Chem. Rev. 105: 793-826).Peptidomimetic Macrocycles of the Invention

In some embodiments of the method, a polypeptide of the inventioncontains one crosslink. In other embodiments of the method, saidpolypeptide contains two cross-links. In some embodiments of the method,one crosslink connects two α-carbon atoms. In other embodiments of themethod, one α-carbon atom to which one crosslink is attached issubstituted with a substituent of formula R—. In another embodiment ofthe method, two α-carbon atoms to which one crosslink is attached aresubstituted with independent substituents of formula R—. In oneembodiment of the methods of the invention, R— is alkyl. For example, R—is methyl. Alternatively, R— and any portion of one crosslink takentogether can form a cyclic structure. In another embodiment of themethod, one crosslink is formed of consecutive carbon-carbon bonds. Forexample, one crosslink may comprise at least 8, 9, 10, 11, or 12consecutive bonds. In other embodiments, one crosslink may comprise atleast 7, 8, 9, 10, or 11 carbon atoms.

In another embodiment of the method, the crosslinked polypeptidecomprises an α-helical domain of a BCL-2 family member. For example, thecrosslinked polypeptide comprises a BH3 domain. In other embodiments,the crosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90%or 95% of any of the sequences in Tables 1, 2, 3 and 4. In someembodiments of the method, the crosslinked polypeptide penetrates cellmembranes by an energy-dependent process and binds to an intracellulartarget.

In some embodiments, said helical polypeptide contains one crosslink. Inother embodiments, said helical polypeptide contains two cross-links.

In some embodiments, one crosslink connects two α-carbon atoms. In otherembodiments, one α-carbon atom to which one crosslink is attached issubstituted with a substituent of formula R—. In another embodiment, twoα-carbon atoms to which one crosslink is attached are substituted withindependent substituents of formula R—. In one embodiment of theinvention, R— is alkyl. For example, R— is methyl. Alternatively, R— andany portion of one crosslink taken together can form a cyclic structure.In another embodiment, one crosslink is formed of consecutivecarbon-carbon bonds. For example, one crosslink may comprise at least 8,9, 10, 11, or 12 consecutive bonds. In other embodiments, one crosslinkmay comprise at least 7, 8, 9, 10, or 11 carbon atoms.

In another embodiment, the crosslinked polypeptide comprises anα-helical domain of a BCL-2 family member. For example, the crosslinkedpolypeptide comprises a BH3 domain. In other embodiments, thecrosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90% or95% of any of the sequences in Tables 1, 2, 3 and 4. In someembodiments, the crosslinked polypeptide penetrates cell membranes by anenergy-dependent process and binds to an intracellular target.

In some embodiments, the peptidomimetic macrocycles of the inventionhave the Formula (I):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

each of v and w is independently an integer from 1-1000;

each of x, y, and z is independently an integer from 0-10; u is aninteger from 1-10; and

n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

In other embodiments, the peptidomimetic macrocycle of Formula (I) is acompound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chainand

is [D]_(v), [E]_(w) as defined above, and n is an integer between 0 and20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In otherembodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In some embodiments, the peptidomimetic macrocycles of the inventionhave the Formula (II):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula

L₁, L₂ and L₃ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

each of v and w is independently an integer from 1-1000;

each of x, y, and z is independently an integer from 0-10; u is aninteger from 1-10; and

n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, the invention provides peptidomimetic macrocyclesof Formula (III):

wherein:each A, C, D, and E is independently a natural or non-natural aminoacid;B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-];R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, unsubstituted or substituted with R₅;L₁, L₂, L₃ and L₄ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene or [—R₄—K—R₄—]n, each being unsubstituted orsubstituted with R₅;K is O, S, SO, SO₂, CO, CO₂, or CONR₃;each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,unsubstituted or substituted with R₅, or part of a cyclic structure witha D residue;R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,unsubstituted or substituted with R₅, or part of a cyclic structure withan E residue; each of v and w is independently an integer from 1-1000;each of x, y, and z is independently an integer from 0-10; u is aninteger from 1-10; andn is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9 or 10. Eachoccurrence of A, B, C, D or E in a macrocycle or macrocycle precursor ofthe invention is independently selected. For example, a sequencerepresented by the formula [A]_(x), when x is 3, encompasses embodimentswhere the amino acids are not identical, e.g. Gln-Asp-Ala as well asembodiments where the amino acids are identical, e.g. Gln-Gln-Gln. Thisapplies for any value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker[-L₁-S-L₂-S-L₃-] as measured from a first Cα to a second Cα is selectedto stabilize a desired secondary peptide structure, such as an α-helixformed by residues of the peptidomimetic macrocycle including, but notnecessarily limited to, those between the first Cα to a second Cα.

Macrocycles or macrocycle precursors are synthesized, for example, bysolution phase or solid-phase methods, and can contain bothnaturally-occurring and non-naturally-occurring amino acids. See, forexample, Hunt, “The Non-Protein Amino Acids” in Chemistry andBiochemistry of the Amino Acids, edited by G. C. Barrett, Chapman andHall, 1985. In some embodiments, the thiol moieties are the side chainsof the amino acid residues L-cysteine, D-cysteine, α-methyl-L cysteine,α-methyl-D-cysteine, L-homocysteine, D-homocysteine,α-methyl-L-homocysteine or α-methyl-D-homocysteine. A bis-alkylatingreagent is of the general formula X-L₂-Y wherein L₂ is a linker moietyand X and Y are leaving groups that are displaced by —SH moieties toform bonds with L₂. In some embodiments, X and Y are halogens such as I,Br, or Cl.

In other embodiments, D and/or E in the compound of Formula I, II or IIIare further modified in order to facilitate cellular uptake. In someembodiments, lipidating or PEGylating a peptidomimetic macrocyclefacilitates cellular uptake, increases bioavailability, increases bloodcirculation, alters pharmacokinetics, decreases immunogenicity and/ordecreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound ofFormula I, II or III represents a moiety comprising an additionalmacrocycle-forming linker such that the peptidomimetic macrocyclecomprises at least two macrocycle-forming linkers. In a specificembodiment, a peptidomimetic macrocycle comprises two macrocycle-forminglinkers.

In the peptidomimetic macrocycles of the invention, any of themacrocycle-forming linkers described herein may be used in anycombination with any of the sequences shown in Tables 1-4 and also withany of the R— substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif. For example, A, B and/or C in the compound of FormulaI, II or III include one or more α-helices. As a general matter,α-helices include between 3 and 4 amino acid residues per turn. In someembodiments, the α-helix of the peptidomimetic macrocycle includes 1 to5 turns and, therefore, 3 to 20 amino acid residues. In specificembodiments, the α-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or5 turns. In some embodiments, the macrocycle-forming linker stabilizesan α-helix motif included within the peptidomimetic macrocycle. Thus, insome embodiments, the length of the macrocycle-forming linker L from afirst Cα to a second Cα is selected to increase the stability of anα-helix. In some embodiments, the macrocycle-forming linker spans from 1turn to 5 turns of the α-helix. In some embodiments, themacrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns,4 turns, or 5 turns of the α-helix. In some embodiments, the length ofthe macrocycle-forming linker is approximately 5 Å to 9 Å per turn ofthe α-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Wherethe macrocycle-forming linker spans approximately 1 turn of an α-helix,the length is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In other embodiments, the invention provides peptidomimetic macrocyclesof Formula (IV) or (IVa):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v is an integer from 1-1000;

w is an integer from 1-1000;

x is an integer from 0-10;

y is an integer from 0-10;

z is an integer from 0-10; and

n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A], when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, the invention provides peptidomimetic macrocyclesof Formula (V):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁, R₂ and R₈ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

w is an integer from 1-1000;

x is an integer from 0-10;

y is an integer from 0-10;

z is an integer from 0-10; and

n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In one embodiment, R₁is H and R₂ is methyl. In another embodiment, R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

Exemplary embodiments of the peptidomimetic macrocycles (SEQ ID NOS89-96, respectively, in order of appearance) are shown below.

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles of the invention may be prepared by any of avariety of methods known in the art. For example, any of the residuesindicated by “X” in Tables 1, 2, 3 or 4 may be substituted with aresidue capable of forming a crosslinker with a second residue in thesame molecule or a precursor of such a residue.

Various methods to effect formation of peptidomimetic macrocycles areknown in the art. For example, the preparation of peptidomimeticmacrocycles of Formula I is described in Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafineister & Verdin, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); andU.S. Pat. No. 7,192,713. The α,α-disubstituted amino acids and aminoacid precursors disclosed in the cited references may be employed insynthesis of the peptidomimetic macrocycle precursor polypeptides.Following incorporation of such amino acids into precursor polypeptides,the terminal olefins are reacted with a metathesis catalyst, leading tothe formation of the peptidomimetic macrocycle.

In other embodiments, the peptidomimetic macrocyles of the invention areof Formula IV or IVa. Methods for the preparation of such macrocyclesare described, for example, in U.S. Pat. No. 7,202,332.

In some embodiments, the synthesis of these peptidomimetic macrocyclesinvolves a multi-step process that features the synthesis of apeptidomimetic precursor containing an azide moiety and an alkynemoiety; followed by contacting the peptidomimetic precursor with amacrocyclization reagent to generate a triazole-linked peptidomimeticmacrocycle. Macrocycles or macrocycle precursors are synthesized, forexample, by solution phase or solid-phase methods, and can contain bothnaturally-occurring and non-naturally-occurring amino acids. See, forexample, Hunt, “The Non-Protein Amino Acids” in Chemistry andBiochemistry of the Amino Acids, edited by G. C. Barrett, Chapman andHall, 1985.

In some embodiments, an azide is linked to the α-carbon of a residue andan alkyne is attached to the α-carbon of another residue. In someembodiments, the azide moieties are azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine,L-ornithine, D-ornithine, alpha-methyl-L-ornithine oralpha-methyl-D-ornithine. In another embodiment, the alkyne moiety isL-propargylglycine. In yet other embodiments, the alkyne moiety is anamino acid selected from the group consisting of L-propargylglycine,D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,(R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoicacid, (R)-2-amino-2-methyl-5-hexynoic acid,(S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoicacid, (S)-2-amino-2-methyl-7-octynoic acid,(R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoicacid and (R)-2-amino-2-methyl-8-nonynoic acid.

In some embodiments, the invention provides a method for synthesizing apeptidomimetic macrocycle, the method comprising the steps of contactinga peptidomimetic precursor of Formula VI or Formula VII:

with a macrocyclization reagent;

wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, L₁ and L₂ are asdefined for Formula (II); R₁₂ is —H when the macrocyclization reagent isa Cu reagent and R₁₂ is —H or alkyl when the macrocyclization reagent isa Ru reagent; and further wherein said contacting step results in acovalent linkage being formed between the alkyne and azide moiety inFormula III or Formula IV. For example, R₁₂ may be methyl when themacrocyclization reagent is a Ru reagent.

In the peptidomimetic macrocycles of the invention, at least one of R₁and R₂ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, unsubstituted or substituted withhalo-. In some embodiments, both R₁ and R₂ are independently alkyl,alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl,or heterocycloalkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of A, B, C, D or E is an α,α-disubstitutedamino acid. In one example, B is an α,α-disubstituted amino acid. Forinstance, at least one of A, B, C, D or E is 2-aminoisobutyric acid.

For example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl. The macrocyclization reagent may be a Cu reagentor a Ru reagent.

In some embodiments, the peptidomimetic precursor is purified prior tothe contacting step. In other embodiments, the peptidomimetic macrocycleis purified after the contacting step. In still other embodiments, thepeptidomimetic macrocycle is refolded after the contacting step. Themethod may be performed in solution, or, alternatively, the method maybe performed on a solid support.

Also envisioned herein is performing the method of the invention in thepresence of a target macromolecule that binds to the peptidomimeticprecursor or peptidomimetic macrocycle under conditions that favor saidbinding. In some embodiments, the method is performed in the presence ofa target macromolecule that binds preferentially to the peptidomimeticprecursor or peptidomimetic macrocycle under conditions that favor saidbinding. The method may also be applied to synthesize a library ofpeptidomimetic macrocycles.

In some embodiments, the alkyne moiety of the peptidomimetic precursorof Formula VI or Formula VII is a sidechain of an amino acid selectedfrom the group consisting of L-propargylglycine, D-propargylglycine,(S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoicacid, (S)-2-amino-2-methyl-5-hexynoic acid,(R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoicacid, (R)-2-amino-2-methyl-6-heptynoic acid,(S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoicacid, (S)-2-amino-2-methyl-8-nonynoic acid, and(R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, the azidemoiety of the peptidomimetic precursor of Formula VI or Formula VII is asidechain of an amino acid selected from the group consisting ofε-azido-L-lysine, ε-azido-D-lysine, ε-azido-α-methyl-L-lysine,ε-azido-α-methyl-D-lysine, δ-azido-α-methyl-L-ornithine, andδ-azido-α-methyl-D-ornithine.

In some embodiments, x+y+z is 3, and A, B and C are independentlynatural or non-natural amino acids. In other embodiments, x+y+z is 6,and A, B and C are independently natural or non-natural amino acids.

In some embodiments of peptidomimetic macrocycles of the invention,[D]_(v) and/or [E]_(w) comprise additional peptidomimetic macrocycles ormacrocyclic structures. For example, [D]_(v) may have the formula:

wherein each A, C, D′, and E′ is independently a natural or non-naturalamino acid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁, R₂ and R₈ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v is an integer from 1-1000;

w is an integer from 1-1000; and

x is an integer from 0-10.

In another embodiment, [E]_(w) has the formula:

wherein the substituents are as defined in the preceding paragraph.

In some embodiments, the contacting step is performed in a solventselected from the group consisting of protic solvent, aqueous solvent,organic solvent, and mixtures thereof. For example, the solvent may bechosen from the group consisting of H₂O, THF, THF/H₂O, tBuOH/H₂O, DMF,DIPEA, CH₃CN or CH₂Cl₂, ClCH₂CH₂Cl or a mixture thereof. The solvent maybe a solvent which favors helix formation.

Alternative but equivalent protecting groups, leaving groups or reagentsare substituted, and certain of the synthetic steps are performed inalternative sequences or orders to produce the desired compounds.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein include, for example, those such as described inLarock, Comprehensive Organic Transformations, VCH Publishers (1989);Greene and Wuts, Protective Groups in Organic Synthesis, 2d. Ed., JohnWiley and Sons (1991); Fieser and Fieser, Fieser and Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The peptidomimetic macrocycles of the invention are made, for example,by chemical synthesis methods, such as described in Fields et al.,Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H.Freeman & Co., New York, N.Y., 1992, p. 77. Hence, for example, peptidesare synthesized using the automated Merrifield techniques of solid phasesynthesis with the amine protected by either tBoc or Fmoc chemistryusing side chain protected amino acids on, for example, an automatedpeptide synthesizer (e.g., Applied Biosystems (Foster City, Calif.),Model 430A, 431, or 433).

One manner of producing the peptidomimetic precursors and peptidomimeticmacrocycles described herein uses solid phase peptide synthesis (SPPS).The C-terminal amino acid is attached to a cross-linked polystyreneresin via an acid labile bond with a linker molecule. This resin isinsoluble in the solvents used for synthesis, making it relativelysimple and fast to wash away excess reagents and by-products. TheN-terminus is protected with the Fmoc group, which is stable in acid,but removable by base. Side chain functional groups are protected asnecessary with base stable, acid labile groups.

Longer peptidomimetic precursors are produced, for example, byconjoining individual synthetic peptides using native chemical ligation.Alternatively, the longer synthetic peptides are biosynthesized by wellknown recombinant DNA and protein expression techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a peptidomimetic precursor of this invention,the amino acid sequence is reverse translated to obtain a nucleic acidsequence encoding the amino acid sequence, preferably with codons thatare optimum for the organism in which the gene is to be expressed. Next,a synthetic gene is made, typically by synthesizing oligonucleotideswhich encode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods.

The peptidomimetic precursors are made, for example, in ahigh-throughput, combinatorial fashion using, for example, ahigh-throughput polychannel combinatorial synthesizer (e.g., ThuramedTETRAS multichannel peptide synthesizer from CreoSalus, Louisville, Ky.or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc.,Louisville, Ky.).

The following synthetic schemes are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein. To simplify the drawings, theillustrative schemes depict azido amino acid analogsε-azido-α-methyl-L-lysine and ε-azido-α-methyl-D-lysine, and alkyneamino acid analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoicacid, and (S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the followingsynthetic schemes, each R₁, R₂, R₇ and R₈ is —H; each L₁ is —(CH₂)₄—;and each L₂ is —(CH₂)—. However, as noted throughout the detaileddescription above, many other amino acid analogs can be employed inwhich R₁, R₂, R₇, R₈, L₁ and L₂ can be independently selected from thevarious structures disclosed herein.

Synthetic Scheme 1 describes the preparation of several compounds of theinvention. Ni(II) complexes of Schiff bases derived from the chiralauxiliary (S)-2-[N—(N′-benzylprolyl)amino]benzophenone (BPB) and aminoacids such as glycine or alanine are prepared as described in Belokon etal. (1998), Tetrahedron Asymm. 9:4249-4252. The resulting complexes aresubsequently reacted with alkylating reagents comprising an azido oralkynyl moiety to yield enantiomerically enriched compounds of theinvention. If desired, the resulting compounds can be protected for usein peptide synthesis.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 2, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aCu(I) in organic or aqueous solutions (Rostovtsev et al. (2002), Angew.Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J. Org. Chem.67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc. 125:11782-11783;Punna et al. (2005), Angew. Chem. Int. Ed 44:2215-2220). In oneembodiment, the triazole forming reaction is performed under conditionsthat favor α-helix formation. In one embodiment, the macrocyclizationstep is performed in a solvent chosen from the group consisting of H₂O,THF, CH₃CN, DMF, DIPEA, tBuOH or a mixture thereof. In anotherembodiment, the macrocyclization step is performed in DMF. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 3, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al.(2002), Angew. Chem. Int. Ed 41:2596-2599; Tornoe et al. (2002), J. Org.Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed44:2215-2220). The resultant triazole-containing peptidomimeticmacrocycle is then deprotected and cleaved from the solid-phase resin bystandard conditions (e.g., strong acid such as 95% TFA). In someembodiments, the macrocyclization step is performed in a solvent chosenfrom the group consisting of CH₂Cl₂, ClCH₂CH₂Cl, DMF, THF, NMP, DIPEA,2,6-lutidine, pyridine, DMSO, H₂O or a mixture thereof. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 4, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aRu(II) reagents, for example Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen etal. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem.Soc. 127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of DMF, CH₃CNand THF.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 5, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Ru(II) reagent on the resin as a crude mixture. For example, thereagent can be Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen et al. (2007),Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem. Soc.127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of CH₂Cl₂,ClCH₂CH₂Cl, CH₃CN, DMF, and THF.

Several exemplary peptidomimetic macrocycles (SEQ ID NOS 97-108,respectively, in order of appearance) are shown in Table 5. “Nle”represents norleucine and replaces a methionine residue. It isenvisioned that similar linkers are used to synthesize peptidomimeticmacrocycles based on the polypeptide sequences disclosed in Table 1through Table 4.

TABLE 5

MW = 2464

MW = 2464

MW= 2478

MW = 2478

MW= 2492

MW = 2492

MW= 2464

MW= 2464

MW = 2478

MW= 2478

MW = 2492

MW = 2492

-   -   Table 5 shows exemplary peptidommimetic macrocycles of the        invention. “Nle” represents norleucine.

The present invention contemplates the use of non-naturally-occurringamino acids and amino acid analogs in the synthesis of thepeptidomimetic macrocycles described herein. Any amino acid or aminoacid analog amenable to the synthetic methods employed for the synthesisof stable triazole containing peptidomimetic macrocycles can be used inthe present invention. For example, L-propargylglycine is contemplatedas a useful amino acid in the present invention. However, otheralkyne-containing amino acids that contain a different amino acid sidechain are also useful in the invention. For example, L-propargylglycinecontains one methylene unit between the α-carbon of the amino acid andthe alkyne of the amino acid side chain. The invention also contemplatesthe use of amino acids with multiple methylene units between theα-carbon and the alkyne. Also, the azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, and alpha-methyl-D-lysine arecontemplated as useful amino acids in the present invention. However,other terminal azide amino acids that contain a different amino acidside chain are also useful in the invention. For example, theazido-analog of L-lysine contains four methylene units between theα-carbon of the amino acid and the terminal azide of the amino acid sidechain. The invention also contemplates the use of amino acids with fewerthan or greater than four methylene units between the α-carbon and theterminal azide. Table 6 shows some amino acids useful in the preparationof peptidomimetic macrocycles of the invention.

TABLE 6

-   -   Table 6 shows exemplary amino acids useful in the preparation of        peptidomimetic macrocycles of the invention.

In some embodiments the amino acids and amino acid analogs are of theD-configuration. In other embodiments they are of the L-configuration.In some embodiments, some of the amino acids and amino acid analogscontained in the peptidomimetic are of the D-configuration while some ofthe amino acids and amino acid analogs are of the L-configuration. Insome embodiments the amino acid analogs are α,α-disubstituted, such asα-methyl-L-propargylglycine, α-methyl-D-propargylglycine,ε-azido-alpha-methyl-L-lysine, and ε-azido-alpha-methyl-D-lysine. Insome embodiments the amino acid analogs are N-alkylated, e.g.,N-methyl-L-propargylglycine, N-methyl-D-propargylglycine,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine.

In some embodiments, the —NH moiety of the amino acid is protected usinga protecting group, including without limitation -Fmoc and -Boc. Inother embodiments, the amino acid is not protected prior to synthesis ofthe peptidomimetic macrocycle.

In other embodiments, peptidomimetic macrocycles of Formula III aresynthesized. The following synthetic schemes describe the preparation ofsuch compounds. To simplify the drawings, the illustrative schemesdepict amino acid analogs derived from L- or D-cysteine, in which L₁ andL₃ are both —(CH₂)—. However, as noted throughout the detaileddescription above, many other amino acid analogs can be employed inwhich L₁ and L₃ can be independently selected from the variousstructures disclosed herein. The symbols “[AA]_(m)”, “[AA]_(n)”,“[AA]_(o)” represent a sequence of amide bond-linked moieties such asnatural or unnatural amino acids. As described previously, eachoccurrence of “AA” is independent of any other occurrence of “AA”, and aformula such as “[AA]_(m)” encompasses, for example, sequences ofnon-identical amino acids as well as sequences of identical amino acids.

In Scheme 6, the peptidomimetic precursor contains two —SH moieties andis synthesized by solid-phase peptide synthesis (SPPS) usingcommercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-trityl-L-cysteine or N-α-Fmoc-S-trityl-D-cysteine.Alpha-methylated versions of D-cysteine or L-cysteine are generated byknown methods (Seebach et al. (1996), Angew. Chem. Int. Ed. Engl.35:2708-2748, and references therein) and then converted to theappropriately protected N-α-Fmoc-S-trityl monomers by known methods(“Bioorganic Chemistry: Peptides and Proteins”, Oxford University Press,New York: 1998, the entire contents of which are incorporated herein byreference). The precursor peptidomimetic is then deprotected and cleavedfrom the solid-phase resin by standard conditions (e.g., strong acidsuch as 95% TFA). The precursor peptidomimetic is reacted as a crudemixture or is purified prior to reaction with X-L₂-Y in organic oraqueous solutions. In some embodiments the alkylation reaction isperformed under dilute conditions (i.e. 0.15 mmol/L) to favormacrocyclization and to avoid polymerization. In some embodiments, thealkylation reaction is performed in organic solutions such as liquid NH₃(Mosberg et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al.(1992), Int. J. Peptide Protein Res. 40:233-242), NH₃/MeOH, or NH₃/DMF(Or et al. (1991), J. Org. Chem. 56:3146-3149). In other embodiments,the alkylation is performed in an aqueous solution such as 6Mguanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.(20):2552-2554). In other embodiments, the solvent used for thealkylation reaction is DMF or dichloroethane

In Scheme 7, the precursor peptidomimetic contains two or more —SHmoieties, of which two are specially protected to allow their selectivedeprotection and subsequent alkylation for macrocycle formation. Theprecursor peptidomimetic is synthesized by solid-phase peptide synthesis(SPPS) using commercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-p-methoxytrityl-L-cysteine orN-α-Fmoc-S-p-methoxytrityl-D-cysteine. Alpha-methylated versions ofD-cysteine or L-cysteine are generated by known methods (Seebach et al.(1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and referencestherein) and then converted to the appropriately protectedN-α-Fmoc-S-p-methoxytrityl monomers by known methods (BioorganicChemistry: Peptides and Proteins, Oxford University Press, New York:1998, the entire contents of which are incorporated herein byreference). The Mmt protecting groups of the peptidomimetic precursorare then selectively cleaved by standard conditions (e.g., mild acidsuch as 1% TFA in DCM). The precursor peptidomimetic is then reacted onthe resin with X-L₂-Y in an organic solution. For example, the reactiontakes place in the presence of a hindered base such asdiisopropylethylamine. In some embodiments, the alkylation reaction isperformed in organic solutions such as liquid NH₃ (Mosberg et al.(1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J.Peptide Protein Res. 40:233-242), NH₃/MeOH or NH₃/DMF (Or et al. (1991),J. Org. Chem. 56:3146-3149). In other embodiments, the alkylationreaction is performed in DMF or dichloroethane. The peptidomimeticmacrocycle is then deprotected and cleaved from the solid-phase resin bystandard conditions (e.g., strong acid such as 95% TFA).

In Scheme 8, the peptidomimetic precursor contains two or more —SHmoieties, of which two are specially protected to allow their selectivedeprotection and subsequent alkylation for macrocycle formation. Thepeptidomimetic precursor is synthesized by solid-phase peptide synthesis(SPPS) using commercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-p-methoxytrityl-L-cysteine,N-α-Fmoc-S-p-methoxytrityl-D-cysteine, N-α-Fmoc-S—S-t-butyl-L-cysteine,and N-α-Fmoc-S—S-t-butyl-D-cysteine. Alpha-methylated versions ofD-cysteine or L-cysteine are generated by known methods (Seebach et al.(1996), Angew. Chem. Int. Ed Engl. 35:2708-2748, and references therein)and then converted to the appropriately protectedN-α-Fmoc-S-p-methoxytrityl or N-α-Fmoc-S—S-t-butyl monomers by knownmethods (Bioorganic Chemistry: Peptides and Proteins, Oxford UniversityPress, New York: 1998, the entire contents of which are incorporatedherein by reference). The S—S-tButyl protecting group of thepeptidomimetic precursor is selectively cleaved by known conditions(e.g., 20% 2-mercaptoethanol in DMF, reference: Galande et al. (2005),J. Comb. Chem. 7:174-177). The precursor peptidomimetic is then reactedon the resin with a molar excess of X-L₂-Y in an organic solution. Forexample, the reaction takes place in the presence of a hindered basesuch as diisopropylethylamine. The Mmt protecting group of thepeptidomimetic precursor is then selectively cleaved by standardconditions (e.g., mild acid such as 1% TFA in DCM). The peptidomimeticprecursor is then cyclized on the resin by treatment with a hinderedbase in organic solutions. In some embodiments, the alkylation reactionis performed in organic solutions such as NH₃/MeOH or NH₃/DMF (Or et al.(1991), J. Org. Chem. 56:3146-3149). The peptidomimetic macrocycle isthen deprotected and cleaved from the solid-phase resin by standardconditions (e.g., strong acid such as 95% TFA).

In Scheme 9, the peptidomimetic precursor contains two L-cysteinemoieties. The peptidomimetic precursor is synthesized by knownbiological expression systems in living cells or by known in vitro,cell-free, expression methods. The precursor peptidomimetic is reactedas a crude mixture or is purified prior to reaction with X-L₂-Y inorganic or aqueous solutions. In some embodiments the alkylationreaction is performed under dilute conditions (i.e. 0.15 mmol/L) tofavor macrocyclization and to avoid polymerization. In some embodiments,the alkylation reaction is performed in organic solutions such as liquidNH₃ (Mosberg et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk etal. (1992), Int. J. Peptide Protein Res. 40:233-242), NH₃/MeOH, orNH₃/DMF (Or et al. (1991), J. Org. Chem. 56:3146-3149). In otherembodiments, the alkylation is performed in an aqueous solution such as6M guanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.(20):2552-2554). In other embodiments, the alkylation is performed inDMF or dichloroethane. In another embodiment, the alkylation isperformed in non-denaturing aqueous solutions, and in yet anotherembodiment the alkylation is performed under conditions that favorα-helical structure formation. In yet another embodiment, the alkylationis performed under conditions that favor the binding of the precursorpeptidomimetic to another protein, so as to induce the formation of thebound α-helical conformation during the alkylation.

Various embodiments for X and Y are envisioned which are suitable forreacting with thiol groups. In general, each X or Y is independently beselected from the general category shown in Table 5. For example, X andY are halides such as —Cl, —Br or —I. Any of the macrocycle-forminglinkers described herein may be used in any combination with any of thesequences shown in Tables 1-4 and also with any of the R— substituentsindicated herein.

TABLE 5 Examples of Reactive Groups Capable of Reacting with ThiolGroups and Resulting Linkages (1) X or Y (2) Resulting Covalent Linkage(3) acrylamide (4) Thioether (5) halide (e.g. (6) Thioether alkyl oraryl halide) (7) sulfonate (8) Thioether (9) aziridine (10) Thioether(11) epoxide (12) Thioether (13) haloacetamide (14) Thioether (15)maleimide (16) Thioether (17) sulfonate (18) Thioether ester

Table 6 shows exemplary macrocycles (SEQ ID NOS 109-114, respectively,in order of appearance) of the invention. “N_(L)” represents norleucineand replaces a methionine residue. It is envisioned that similar linkersare used to synthesize peptidomimetic macrocycles based on thepolypeptide sequences disclosed in Table 1 through Table 4.

TABLE 6 Examples of Peptidomimetic Macrocycles of the Invention

MW = 2477

MW = 2463

MW = 2525

MW = 2531

MW = 2475

MW = 2475

For the examples shown in this table, “N_(L)” represents norleucine.

The present invention contemplates the use of both naturally-occurringand non-naturally-occurring amino acids and amino acid analogs in thesynthesis of the peptidomimetic macrocycles of Formula (III). Any aminoacid or amino acid analog amenable to the synthetic methods employed forthe synthesis of stable bis-sulfhydryl containing peptidomimeticmacrocycles can be used in the present invention. For example, cysteineis contemplated as a useful amino acid in the present invention.However, sulfur containing amino acids other than cysteine that containa different amino acid side chain are also useful. For example, cysteinecontains one methylene unit between the α-carbon of the amino acid andthe terminal —SH of the amino acid side chain. The invention alsocontemplates the use of amino acids with multiple methylene unitsbetween the α-carbon and the terminal —SH. Non-limiting examples includeα-methyl-L-homocysteine and α-methyl-D-homocysteine. In some embodimentsthe amino acids and amino acid analogs are of the D-configuration. Inother embodiments they are of the L-configuration. In some embodiments,some of the amino acids and amino acid analogs contained in thepeptidomimetic are of the D-configuration while some of the amino acidsand amino acid analogs are of the L-configuration. In some embodimentsthe amino acid analogs are α,α-disubstituted, such asα-methyl-L-cysteine and α-methyl-D-cysteine.

The invention includes macrocycles in which macrocycle-forming linkersare used to link two or more —SH moieties in the peptidomimeticprecursors to form the peptidomimetic macrocycles of the invention. Asdescribed above, the macrocycle-forming linkers impart conformationalrigidity, increased metabolic stability and/or increased cellpenetrability. Furthermore, in some embodiments, the macrocycle-forminglinkages stabilize the α-helical secondary structure of thepeptidomimetic macrocyles. The macrocycle-forming linkers are of theformula X-L₂-Y, wherein both X and Y are the same or different moieties,as defined above. Both X and Y have the chemical characteristics thatallow one macrocycle-forming linker -L₂- to bis alkylate thebis-sulfhydryl containing peptidomimetic precursor. As defined above,the linker -L₂- includes alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, orheterocycloarylene, or —R₄—K—R₄—, all of which can be optionallysubstituted with an R₅ group, as defined above. Furthermore, one tothree carbon atoms within the macrocycle-forming linkers -L₂-, otherthan the carbons attached to the —SH of the sulfhydryl containing aminoacid, are optionally substituted with a heteroatom such as N, S or O.

The L₂ component of the macrocycle-forming linker X-L₂-Y may be variedin length depending on, among other things, the distance between thepositions of the two amino acid analogs used to form the peptidomimeticmacrocycle. Furthermore, as the lengths of L₁ and/or L₃ components ofthe macrocycle-forming linker are varied, the length of L₂ can also bevaried in order to create a linker of appropriate overall length forforming a stable peptidomimetic macrocycle. For example, if the aminoacid analogs used are varied by adding an additional methylene unit toeach of L₁ and L₃, the length of L₂ are decreased in length by theequivalent of approximately two methylene units to compensate for theincreased lengths of L₁ and L₃.

In some embodiments, L₂ is an alkylene group of the formula —(CH₂)_(n)—,where n is an integer between about 1 and about 15. For example, n is 1,2, 3, 4, 5, 6, 7, 8, 9 or 10. In other embodiments, L₂ is an alkenylenegroup. In still other embodiments, L₂ is an aryl group.

Table 7 shows additional embodiments of X-L₂-Y groups.

TABLE 7 Exemplary X—L₂—Y groups of the invention.

Each X and Y in this table, is, for example, independently Cl—, Br— orI—.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 10, the peptidomimetic precursor contains atleast one olefin moiety and an alkyne moiety and is synthesized bysolution-phase or solid-phase peptide synthesis (SPPS) usingcommercially available N-a-Fmoc-protected amino acids and theN-α-Fmoc-protected forms of the amino acid(S)-2-amino-2-methyl-6-heptenoic acid. The free N-terminus of thepeptide is acylated with 6-heptenoic acid standard amide bond formingconditions. The terminal olefin of the acylating group is crosslinked tothe terminal olefin of the internal amino acid sidechain using rutheniumcatalyzed olefin metathesis as described in U.S. Pat. No. 5,811,515. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA).

Additional methods of forming peptidomimetic macrocycles which areenvisioned as suitable to perform the present invention include thosedisclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem. (2003), 68,pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat. No. 5,446,128; U.S. Pat.No. 5,824,483; U.S. Pat. No. 6,713,280; and U.S. Pat. No. 7,202,332. Insuch embodiments, amino acid precursors are used containing anadditional substituent R— at the alpha position. Such aminoacids areincorporated into the macrocycle precursor at the desired positions,which may be at the positions where the crosslinker is substituted or,alternatively, elsewhere in the sequence of the macrocycle precursor.Cyclization of the precursor is then effected according to the indicatedmethod.

Assays

The properties of the peptidomimetic macrocycles of the invention areassayed, for example, by using the methods described below.

Assay to Determine α-helicity.

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, unmodified pro-apoptotic BH3 domains are predominantlyrandom coils in solution, with α-helical content usually under 25%.Peptidomimetic macrocycles with optimized linkers, on the other hand,possess, for example, an alpha-helicity that is at least two-foldgreater than that of a corresponding uncrosslinked polypeptide. In someembodiments, macrocycles of the invention will possess an alpha-helicityof greater than 50%. To assay the helicity of peptidomimetic macrocylesof the invention, such as BH3 domain-based macrocycles, the compoundsare dissolved in an aqueous solution (e.g. 50 mM potassium phosphatesolution at pH 7, or distilled H₂O, to concentrations of 25-50 μM).Circular dichroism (CD) spectra are obtained on a spectropolarimeter(e.g., Jasco J-710) using standard measurement parameters (e.g.temperature, 20° C.; wavelength, 190-260 nm; step resolution, 0.5 nm;speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm;path length, 0.1 cm). The α-helical content of each peptide iscalculated by dividing the mean residue ellipticity (e.g. [Φ]222obs) bythe reported value for a model helical decapeptide (Yang et al. (1986),Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding uncrosslinked polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions. To assaythe effect of macrocycle formation on meltine temperature,peptidomimetic macrocycles or unmodified peptides are dissolved indistilled H₂O (e.g. at a final concentration of 50 μM) and the Tm isdetermined by measuring the change in ellipticity over a temperaturerange (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710)using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention may besubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding uncrosslinked polypeptide.For example, the peptidomimetic macrocycle and a correspondinguncrosslinked polypeptide are incubated with trypsin agarose and thereactions quenched at various time points by centrifugation andsubsequent HPLC injection to quantitate the residual substrate byultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycleand peptidomimetic precursor (5 mcg) are incubated with trypsin agarose(Pierce) (S/E ˜125) for 0, 10, 20, 90, and 180 minutes. Reactions arequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant is quantified by HPLC-based peak detectionat 280 nm. The proteolytic reaction displays first order kinetics andthe rate constant, k, is determined from a plot of ln [S] versus time(k=−1×slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding uncrosslinked polypeptide, and possess an ex vivohalf-life of 12 hours or more. For ex vivo serum stability studies, avariety of assays may be used. For example, a peptidomimetic macrocycleand/or a corresponding uncrosslinked polypeptide (2 mcg) are eachincubated with fresh mouse, rat and/or human serum (e.g. 1-2 mL) at 37°C. for 0, 1, 2, 4, 8, and 24 hours. Samples of differing macrocycleconcentration may be prepared by serial dilution with serum. Todetermine the level of intact compound, the following procedure may beused: The samples are extracted by transferring 100 μl of sera to 2 mlcentrifuge tubes followed by the addition of 10 μL of 50% formic acidand 500 μL acetonitrile and centrifugation at 14,000 RPM for 10 min at4±2° C. The supernatants are then transferred to fresh 2 ml tubes andevaporated on Turbovap under N₂<10 psi, 37° C. The samples arereconstituted in 100 μL of 50:50 acetonitrile:water and submitted toLC-MS/MS analysis. Equivalent or similar procedures for testing ex vivostability are known and may be used to determine stability ofmacrocycles in serum.

In Vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) issued, for example. The FPA technique measuresthe molecular orientation and mobility using polarized light andfluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuesmay be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle of the invention shows, in some instances,similar or lower Kd than a corresponding uncrosslinked polypeptide.

Acceptor proteins for BH3-peptides such as BCL-2, BCL-X_(L), BAX or MCL1may, for example, be used in this assay. Acceptor proteins for p53peptides such as MDM2 or MDMX may also be used in this assay.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide (e.g. a BH3 peptide or a p53 peptide) andan acceptor protein, a fluorescence polarization assay (FPA) utilizing afluoresceinated peptidomimetic macrocycle derived from a peptidomimeticprecursor sequence is used, for example. The FPA technique measures themolecular orientation and mobility using polarized light and fluorescenttracer. When excited with polarized light, fluorescent tracers (e.g.,FITC) attached to molecules with high apparent molecular weights (e.g.FITC-labeled peptides bound to a large protein) emit higher levels ofpolarized fluorescence due to their slower rates of rotation as comparedto fluorescent tracers attached to smaller molecules (e.g. FITC-labeledpeptides that are free in solution). A compound that antagonizes theinteraction between the fluoresceinated peptidomimetic macrocycle and anacceptor protein will be detected in a competitive binding FPAexperiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay. Acceptor proteins for BH3-peptides such as BCL2, BCL-XL, BAXor MCL1 can be used in this assay. Acceptor proteins for p53 peptidessuch as MDM2 or MDMX can be used in this assay.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle, includingBCL2, MCL1, BCL-XL, A1, BAX, BAK, MDM2 or MDMX.

Cellular Penetrability Assays.

A peptidomimetic macrocycle is, for example, more cell permeablecompared to a corresponding uncrosslinked polypeptide. In someembodiments, the peptidomimetic macrocycles are more cell permeable thana corresponding uncrosslinked polypeptides. Peptidomimetic macrocycleswith optimized linkers possess, for example, cell penetrability that isat least two-fold greater than a corresponding uncrosslinkedpolypeptide, and often 20% or more of the applied peptidomimeticmacrocycle will be observed to have penetrated the cell after 4 hours.To measure the cell penetrability of peptidomimetic macrocycles andcorresponding uncrosslinked polypeptides, intact cells are incubatedwith fluoresceinated peptidomimetic macrocycles or correspondinguncrosslinked polypeptides (10 μM) for 4 hrs in serum free media at 37°C., washed twice with media and incubated with trypsin (0.25%) for 10min at 37° C. The cells are washed again and resuspended in PBS.Cellular fluorescence is analyzed, for example, by using either aFACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.

Cellular Efficacy Assays.

The efficacy of certain peptidomimetic macrocycles is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) toidentify those that kill at EC50<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the peptidomimetic macrocycles. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the peptidomimetic macrocycles killcells by activating the apoptotic machinery. For example, the CellTiter-glo assay is used which determines cell viability as a function ofintracellular ATP concentration.

In Vivo Stability Assay.

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rats by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 μL offresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

To determine the anti-oncogenic activity of peptidomimetic macrocyclesof the invention in vivo, the compounds are, for example, given alone(IP, IV, PO, by inhalation or nasal routes) or in combination withsub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,doxorubicin, etoposide). In one example, 5×10⁶ RS4;11 cells (establishedfrom the bone marrow of a patient with acute lymphoblastic leukemia)that stably express luciferase are injected by tail vein in NOD-SCIDmice 3 hrs after they have been subjected to total body irradiation. Ifleft untreated, this form of leukemia is fatal in 3 weeks in this model.The leukemia is readily monitored, for example, by injecting the micewith D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g.,Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton,Mass.). Total body bioluminescence is quantified by integration ofphotonic flux (photons/sec) by Living Image Software (Caliper LifeSciences, Hopkinton, Mass.). Peptidomimetic macrocycles alone or incombination with sub-optimal doses of relevant chemotherapeutics agentsare, for example, administered to leukemic mice (10 days afterinjection/day 1 of experiment, in bioluminescence range of 14-16) bytail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7to 21 days. Optionally, the mice are imaged throughout the experimentevery other day and survival monitored daily for the duration of theexperiment. Expired mice are optionally subjected to necropsy at the endof the experiment. Another animal model is implantation into NOD-SCIDmice of DoHH2, a cell line derived from human follicular lymphoma, thatstably expresses luciferase. These in vivo tests optionally generatepreliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical Trials.

To determine the suitability of the peptidomimetic macrocycles of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with cancer and in need of treatment areselected and separated in treatment and one or more control groups,wherein the treatment group is administered a peptidomimetic macrocycleof the invention, while the control groups receive a placebo or a knownanti-cancer drug. The treatment safety and efficacy of thepeptidomimetic macrocycles of the invention can thus be evaluated byperforming comparisons of the patient groups with respect to factorssuch as survival and quality-of-life. In this example, the patient grouptreated with a peptidomimetic macrocyle show improved long-term survivalcompared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

Methods of administration include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,rectal, by inhalation, or topical by application to ears, nose, eyes, orskin.

The peptidomimetic macrocycles of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the peptidomimetic macrocycles of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution. The term “parenteral” as used hereinrefers modes of administration including intravenous, intraarterial,intramuscular, intraperitoneal, intrasternal, and subcutaneous.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of apeptidomimetic macrocycle and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

Methods of Use

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe p53 MDM2 system, labeled stabilized peptidomimetic macrocyles basedon the p53 is used in an MDM2 binding assay along with small moleculesthat competitively bind to MDM2. Competitive binding studies allow forrapid in vitro evaluation and determination of drug candidates specificfor the p53/MDM2 system. Likewise in the BH3/BCL-X_(L) anti-apoptoticsystem labeled peptidomimetic macrocycles based on BH3 can be used in aBCL-X_(L) binding assay along with small molecules that competitivelybind to BCL-X_(L). Competitive binding studies allow for rapid in vitroevaluation and determination of drug candidates specific for theBH3/BCL-X_(L) system. The invention further provides for the generationof antibodies against the peptidomimetic macrocycles. In someembodiments, these antibodies specifically bind both the peptidomimeticmacrocycle and the p53 or BH3 peptidomimetic precursors upon which thepeptidomimetic macrocycles are derived. Such antibodies, for example,disrupt the p53/MDM2 or BH3/BCL-XL systems, respectively.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) BCL-2 family member expression or activity(e.g., extrinsic or intrinsic apoptotic pathway abnormalities). It isbelieved that some BCL-2 type disorders are caused, at least in part, byan abnormal level of one or more BCL-2 family members (e.g., over orunder expression), or by the presence of one or more BCL-2 familymembers exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of the BCL-2 family member or the enhancement ofthe level and/or activity of the BCL-2 family member, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, the present invention provides methods for treatingor preventing hyperproliferative disease by interfering with theinteraction or binding between p53 and MDM2 in tumor cells. Thesemethods comprise administering an effective amount of a compound of theinvention to a warm blooded animal, including a human, or to tumor cellscontaining wild type p53. In some embodiments, the administration of thecompounds of the present invention induce cell growth arrest orapoptosis. In other or further embodiments, the present invention isused to treat disease and/or tumor cells comprising elevated MDM2levels. Elevated levels of MDM2 as used herein refers to MDM2 levelsgreater than those found in cells containing more than the normal copynumber (2) of mdm2 or above about 10,000 molecules of MDM2 per cell asmeasured by ELISA and similar assays (Picksley et al. (1994), Oncogene9, 2523 2529).

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, the peptidomimetics macrocycles of the invention isused to treat, prevent, and/or diagnose cancers and neoplasticconditions. As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of breast, lung, liver,colon and ovarian origin. “Pathologic hyperproliferative” cells occur indisease states characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the peptidomimeticsmacrocycles are novel therapeutic agents for controlling breast cancer,ovarian cancer, colon cancer, lung cancer, metastasis of such cancersand the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit. Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia

In other or further embodiments, the peptidomimetics macrocycles of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic peptidomimetics macrocycles of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons, and the anti-apoptotic peptidomimetics macrocycles ofthe invention are used, in some embodiments, in the treatment of thesedisorders. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa,spinal muscular atrophy, and various forms of cerebellar degeneration.The cell loss in these diseases does not induce an inflammatoryresponse, and apoptosis appears to be the mechanism of cell death. Inaddition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis. In other orfurther embodiments, the anti-apoptotic peptidomimetics macrocycles ofthe invention are used to treat all such disorders associated withundesirable cell death.

Some examples of immunologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto organ transplant rejection, arthritis, lupus, IBD, Crohn's disease,asthma, multiple sclerosis, diabetes, etc.

Some examples of neurologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto diabetes, hypothyroidism, hypopituitarism, hypoparathyroidism,hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the peptidomimetics macrocycles of theinvention include, but are not limited to, atherosclerosis, myocardialinfarction, stroke, thrombosis, aneurism, heart failure, ischemic heartdisease, angina pectoris, sudden cardiac death, hypertensive heartdisease; non-coronary vessel disease, such as arteriolosclerosis, smallvessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stent, syntheticor natural excision grafts, indwelling catheter, valve or otherimplantable devices. Preferred cardiovascular disorders includeatherosclerosis, myocardial infarction, aneurism, and stroke.

EXAMPLES

The following section provides illustrative examples of the presentinvention.

Example 1 Synthesis of Peptidomimetic Macrocycles of the Invention

α-helical BID, BIM and p53 peptidomimetic macrocycles were synthesized,purified and analyzed as previously described (Walensky et al (2004)Science 305:1466-70; Walensky et al (2006) Mol Cell 24:199-210; Bernalet al (2007) J. Am. Chem. Soc. 9129, 2456-2457) and as indicated below.The macrocycles used in this study are shown in FIG. 1. Thecorresponding uncrosslinked polypeptides represent the naturalcounterparts of the peptidomimetic macrocycles of the invention.

Alpha,alpha-disubstituted non-natural amino acids containing olefinicside chains were synthesized according to Williams et al. (1991) J. Am.Chem. Soc. 113:9276; Schafineister et al. (2000) J. Am. Chem. Soc.122:5891 and Verdine et al PCT WO 2008/121767. Peptidomimeticmacrocycles were designed by replacing two or more naturally occurringamino acids (see FIG. 1) with the corresponding synthetic amino acids.Substitutions were made at the i and i+4 or i and i+7 positions.Macrocycles were generated by solid phase peptide synthesis followed byolefin metathesis-based crosslinking of the synthetic amino acids viatheir olefin-containing side chains.

In the sequences shown, the following abbreviations are used: “Nle”represents norleucine, “Aib” represents 2-aminoisobutyric acid, “Ac”represents acetyl, and “Pr” represents propionyl Amino acids representedas “$” are alpha-Me S5-pentenyl-alanine olefin amino acids connected byan all-carbon i to i+4 crosslinker comprising one double bond Aminoacids represented as “$r5” are alpha-Me R5-pentenyl-alanine olefin aminoacids connected by an all-carbon i to i+4 crosslinker comprising onedouble bond. Amino acids represented as “$s8” are alpha-MeS8-octenyl-alanine olefin amino acids connected by an all-carbon i toi+7 crosslinker comprising one double bond. Amino acids represented as“$r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected by anall-carbon i to i+7 crosslinker comprising one double bond Amino acidsrepresented as “St” connect two all-carbon crosslinkers (S-5/R-5bis-pentenyl amino acids) Amino acids represented as “Hep” areolefin-crosslinked N-terminal heptenoic acids. The crosslinkers arelinear all-carbon crosslinker comprising eight or eleven carbon atomsbetween the alpha carbons of each amino acid.

The non-natural amino acids (R and S enantiomers of the 5-carbonolefinic amino acid and the S enantiomer of the 8-carbon olefinic aminoacid) were characterized by nuclear magnetic resonance (NMR)spectroscopy (Varian Mercury 400) and mass spectrometry (Micromass LCT).Peptide synthesis was performed either manually or on an automatedpeptide synthesizer (Applied Biosystems, model 433A), using solid phaseconditions, rink amide AM resin (Novabiochem), and Fmoc main-chainprotecting group chemistry. For the coupling of natural Fmoc-protectedamino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA wereemployed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. Olefin metathesiswas performed in the solid phase using 10 mM Grubbs catalyst (Blackewellet al. 1994 supra) (Strem Chemicals) dissolved in degasseddichloromethane and reacted for 2 hours at room temperature. Isolationof metathesized compounds was achieved by trifluoroacetic acid-mediateddeprotection and cleavage, ether precipitation to yield the crudeproduct, and high performance liquid chromatography (HPLC) (VarianProStar) on a reverse phase C18 column (Varian) to yield the purecompounds. Chemical composition of the pure products was confirmed byLC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLCsystem) and amino acid analysis (Applied Biosystems, model 420A).

Example 2 Synthesis of N-Terminal Cross-Linked SP-18 & SP19 Macrocycles

(SEQ ID NOS 115-117, respectively, in order of appearance)

The peptides were elongated on a Thuramed Tetras automated multichannelpeptide synthesizer starting with a4-(2′4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyllinked polystyrene resin (Rink AM resin). The amino acids (5 eq) werecoupled using standard solid phase protocols based onfluorenylmethoxycarbonyl (Fmoc) protection and2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU) as the coupling agent (5 eq). Double couplingwas used during the automated process for all of the amino acids exceptfor the α-methylated Fmoc-protected olefinic amino acids which weresingle coupled with longer reaction times. After the final amino acidwas added to the peptide, the Fmoc group was removed and the free aminewas acylated using acetic anhydride in 10% DIEA. The linear peptide wasassembled as above on resin (0.5 mmol based on initial resin loading)incorporating the desired Fmoc-protected olefinic amino acid. After thecoupling of the last amino acid, the N-terminus was acylated with6-heptenoic acid (5 eq) using the method outlined above. The resin waswashed with DCM. The resin was dried under reduced pressure and taken upin an anhydrous DCM solution of Grubbs I catalyst (20 mL, 4 mg/mL, 0.02mmol). After 18 h, the reaction was filtered and the resin was washedwith DCM. The olefin metathesis step was repeated until the startingmaterial was fully consumed. The cyclized peptide was simultaneouslycleaved from the resin and the protecting groups on the sidechainsremoved by treating the resin with a solution (15 mL) of trifluoroaceticacid (TFA) (93.5%), water (2.5%), triisopropylsilane (TIPS), (2.5%), andethanedithiol (EDT) (2.5%). Chilled diethylether (200 mL) was addedafter 4 h. The mixture was centrifuged and the supernatant decanted. Thepellet was suspended in 1:1 acetonitrile/water (50 mL) and lyophilized.The crude peptide was purified using C₁₈ reversed-phase HPLC withacetonitrile and water (with 0.1% TFA) as the mobile phase. Thefractions containing the desired peptide were pooled. The fractions werelyophilized twice in 50:50 acetonitrile:HCl (aq) (60 mN, then 10 mN) andonce in 50:50 acetonitrile:water to give SP18 or SP19 as a colorlesssolid (SP18: 16 mg, SP19: 32 mg).

Example 3 Sample and Standard Curve Preparation

For in vivo plasma stability studies 50 μL of 10 mM of each macrocyclein DMSO was combined with 9950 μL rat plasma (1:200 v/v) and mixed byvortexing (4 minutes). This stock was serially diluted in rat plasma toyield 9 standards (20-20,000, or 100-50,000 ng/mL range). Highconcentration (early time point) test samples were diluted 10:1 or 5:1in blank plasma. All samples, including plasma blank, were combined 1:1v/v with internal standard peptide in plasma.

Example 4 Pharmacokinetic Analysis

The IV dose formulation is prepared by dissolving peptidomimeticmacrocycles of the invention in 5% DMSO/D5W to achieve a 10 mg/Kg/dose.Canulated Crl:CD® (SD) male rats (7-8 weeks old, Charles RiverLaboratories) are used in these studies. Intravenous doses areadministered via the femoral cannula and the animals are dosed at 10mL/kg per single injection. Blood for pharmacokinetic analysis iscollected at 10 time points (0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24hrs post-dose). Animals are terminated (without necropsy) followingtheir final sample collection.

The whole blood samples are centrifuged (˜1500×g) for 10 min at ˜4° C.Plasma is prepared and transferred within 30 min of bloodcollection/centrifugation to fresh tubes that are frozen and stored inthe dark at ˜70° C. until they are prepared for LC-MS/MS analysis.

Sample extraction is achieved, for example, by adding 10 μL of 50%formic acid to 100 μL plasma (samples or stds), following by vortexingfor 10 seconds. 500 μL acetonitrile is added to the followed byvortexing for 2 minutes and centrifuged at 14,000 rpm for 10 minutes at˜4° C. Supernatants are transferred to clean tubes and evaporated onturbovap<10 psi at 37° C. Prior to LC-MS/MS analysis samples arereconstituted with 100 μL of 50:50 acetonitrile:water.

The peak plasma concentration (C_(max)), the time required to achievethe peak plasma concentration (t_(max)), the plasma terminal half-life(t_(1/2)), the area under the plasma concentration time curve (AUC), theclearance and volume of distribution are calculated from the plasmaconcentration data. All pharmacokinetic calculations are done usingWinNonlin version 4.1 (Pharsight Corp) by non-compartmental analysis.Results of this analysis for peptidomimetic macrocycles of the inventionare shown in FIG. 2.

The following LC-MS/MS method is used. In brief, the LC-MS/MSinstruments used was an API 365 (Applied Biosystems). The analyticalcolumn was a Phenomenex Synergi (4μ, Polar-RP, 50 mm×2 mm) and mobilephases A (0.1% formic acid in water) and B (0.1% formic acid inmethanol) are pumped at a flow rate of 0.4 ml/min to achieve thefollowing gradient:

Time (min) % B 0 15 0.5 15 1.5 95 4.5 95 4.6 15 8.0 StopMRM: 814.0 to 374.2 (positive ionization)

Example 5 Determination of Apparent Affinity to Serum Proteins (K_(d)*)

The measurement of apparent Kd values for serum protein by EC₅₀ shiftanalysis provides a simple and rapid means of quantifying the propensityof experimental compounds to bind serum albumin and other serumproteins. A linear relationship exists between the apparent EC′₅₀ in thepresence of serum protein (EC₅₀) and the amount of serum protein addedto an in vitro assay. This relationship is defined by the bindingaffinity of the compound for serum proteins, expressed as K_(d)*. Thisterm is an experimentally determined, apparent dissociation constantthat may result from the cumulative effects of multiple, experimentallyindistinguishable, binding events. The form of this relationship ispresented here in Eq. (1), and its derivation can be found in Copelandet al, Biorg. Med. Chem. Lett. 2004, 14:2309-2312.

$\begin{matrix}{{EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}} & (1)\end{matrix}$

A significant proportion of serum protein binding can be ascribed todrug interactions with serum albumin, due to the very high concentrationof this protein in serum (35-50 g/L or 530-758 μM). To calculate the Kdvalue for these compounds we have assumed that the shift in EC₅₀ uponprotein addition can be ascribed fully to the serum albumin present inthe added serum, where P is 700 μM for 100% serum, P is 70 μM for 10%serum, etc. We further made the simplifying assumption that all of thecompounds bind serum albumin with a 1:1 stoichiometry, so that the termn in Eq. (1) is fixed at unity. With these parameters in place wecalculated the K_(d)* value for each cross-linked polypeptide from thechanges in EC₅₀ values with increasing serum (and serum protein)concentrations by nonlinear regression analysis of Eq. 1 usingMathematica 4.1 (Wolfram Research, Inc., www.wolfram.com). EC′₅₀ valuesin whole blood are estimated by setting P in Eq. 1 to 700 μM [serumalbumin].

The free fraction in blood is estimated per the following equation, asderived by Trainor, Expert Opin. Drug Disc., 2007, 2(1):51-64, where thetotal serum albumin concentration (for example, [HSA]_(total)) is set at700 μM. The formula below may be used with any type of serum albumin,including rat serum albumin.

$\begin{matrix}{{FreeFraction} = \frac{K_{d}^{*}}{K_{d}^{*} + \lbrack{HSA}\rbrack_{total}}} & (2)\end{matrix}$

Example 6 Determination of α-Helicity

Two vials (1.0 mg) of each sample were dissolved in 50% acetonitrile/50%water for a final concentration of 1.0 mg/ml. 100 μL or approximately0.1 mg of each sample was aliquoted into each vial. Three 30 μL sampleswere taken for amino acid analysis. All samples were lyophilizedovernight then stored at −20° C. Samples were diluted to severaldifferent concentrations (1.0 mg/ml, 0.5 mg/ml, 0.1 mg/ml, and 0.05mg/ml) and put into varying path length cells (1.0 mm, 2.0 mm, 5.0 mm,and 10.0 mm). All samples were visually inspected for debris and scanswere taken of each sample at 5° C. to determine ideal path length andconcentration for solubility. The samples were soluble at 0.05 mg/ml in20 mM phosphoric acid pH 2.0 buffer. All scans and temperature meltswere performed in this buffer condition (benign buffer—20 mM phosphoricacid pH 2.0) in a 10.0 mm CD cell. All samples were run on a Jasco J-815spectropolarimeter using the Spectra Manager software package Sampleswere dissolved in 2.0 mL benign buffer (denoted 0% TFE) or buffercontaining 5%, 10%, 15%, 20% or 50% Trifluoroethanol (TFE) fromSigma-Aldrich (catalog T63002). CD scans were run from 250-190 nm at 5°C. Data was collected every 0.2 nm. Appropriate buffer blanks were runbefore each CD scan and the buffer was subtracted from each run.Temperature melts were run from 5° C.-80° C. with reverse melts from 80°C.-5° C. immediately following. Data was collected every 0.2 degreesAmino acid analysis was done using the AccQ Tag System (Waters, Milford,Mass.) on an Agilent 1100 HPLC. Briefly, the peptide aliquots werehydrolyzed by adding 200 uL of 6M HCl to each aliquot and heating thesamples at 110 deg C. for 24 hr. The samples were then vacuum dried andthe resulting residue was resuspended in 200 uL of 200 mM HCl. Using thereagents provided in the AccQ Tag Chemistry Kit, each free amino acid in20 uL of the hydrolysate was derivatized with a quinoline moiety. HPLCwas used to separate the individual amino acids for each hydrolysatesample using a custom gradient and a custom column, and the abundance ofeach was measured by UV at 254 nm. A sodium acetate buffer, pH 5.05 anda 60/40 (v/v) acetonitrile/water were the running buffers. By comparingthe area of each peak to a set of standards with a known amount of eachamino acid, the absolute amounts of each amino acid present in eachhydrolysate sample were determined. Using the sequence of each peptide,the concentration of the peptide was determined using either the amountof alanine or leucine in the sample. All data was imported and saved inExcel files where percent helix, molar ellipticity, or concentrations byAAA were calculated. Percent helicity in aqueous solution was determinedby dividing the molar ellipticity (222 nm) in 0% TFE for eachcrosslinked peptide by the molar ellipticity (222 nm) in 50% TFE for theparent peptide, with the assumption that the parent peptide is 100%helical in 50% TFE.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of screening for an improvedalpha-helical polypeptide with an increased in vivo half-lifecomprising: a. providing a parent alpha-helical polypeptide; b.installing at least one cross-link in the parent alpha-helicalpolypeptide, thereby resulting in a cross-linked polypeptide of Formula(I):

wherein: each A, C, D, and E is independently a natural or non-naturalamino acid; each B is independently a natural or non-natural amino acid,amino acid analog,

 [—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-]; each R₁ and R₂ areindependently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or an additionalcross-link L, unsubstituted or substituted with halo-; each R₃ isindependently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅; L is alkyl, alkenyl oralkynyl; each L₃ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅; each R₄ is independently alkylene, alkenylene,alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene,or heteroarylene; each K is independently O, S, SO, SO₂, CO, CO₂, orCONR₃; each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆,—SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or atherapeutic agent; each R₆ is independently —H, alkyl, alkenyl, alkynyl,arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, aradioisotope or a therapeutic agent; each R₇ is independently —H, alkyl,alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substitutedwith R₅, or part of a cyclic structure with a D residue; each R₈ isindependently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue; each of v and w is independently an integerfrom 1-1000; each of x, y, and z is independently an integer from 0-10;u is an integer of value 1 or more; n is an integer from 1-5; wherein atleast one-crosslink connects two a-carbon atoms; and c. determining anapparent affinity (K_(d)*) of the cross-linked polypeptide; d.determining the in vivo half-life of the cross-linked polypeptiderelative to the parent alpha-helical polypeptide; and e. selecting thecross-linked polypeptide as an improved alpha-helical polypeptide, ifthe K_(d)* of the cross-linked polypeptide is in the range of 1 to 70micromolar and the cross-linked polypeptide has an increased in vivohalf-life relative to a corresponding polypeptide lacking said at leastone cross-link.
 2. The method of claim 1, wherein at least one of R₁ andR₂ is alkyl.
 3. The method of claim 1, wherein at least one of R₁ and R₂is methyl.
 4. The method of claim 1, wherein u is
 2. 5. The method ofclaim 1, wherein one crosslink is formed of consecutive carbon-carbonbonds.
 6. The method of claim 1, wherein one crosslink contains at least8 consecutive bonds.
 7. The method of claim 1, wherein one crosslinkcontains 9 consecutive bonds.
 8. The method of claim 1, wherein onecrosslink contains 12 consecutive bonds.
 9. The method of claim 1,wherein one crosslink comprises at least 7 carbon atoms.
 10. The methodof claim 1, wherein one crosslink comprises at least 10 carbon atoms.11. The method of claim 1, wherein the crosslinked polypeptide comprisesan α-helical domain of a BCL-2 family member.
 12. The method of claim 1,wherein the crosslinked polypeptide comprises a BH3 domain.
 13. Themethod of claim 1, wherein the crosslinked polypeptide has a K_(d)* of 1to 10 micromolar.
 14. The method of claim 1, wherein the crosslinkedpolypeptide has a K_(d)* in the range 10-70 micromolar.
 15. The methodof claim 1, wherein the cross-linked polypeptide is selected such thatthe % helicity of the crosslinked polypeptide is 25% or greater at roomtemperature under aqueous conditions.
 16. The method of claim 1, whereinthe cross-linked polypeptide is selected such that the % helicity of thecrosslinked polypeptide is 50% or greater at room temperature underaqueous conditions.
 17. The method of claim 1, wherein the cross-linkedpolypeptide is selected such that the % helicity of the crosslinkedpolypeptide is 75% or greater at room temperature under aqueousconditions.
 18. The method of claim 1, wherein the in vivo half-life ofsaid polypeptide is determined after intravenous administration.